Rada J A, Cornuet P K, Hassell J R
Department of Ophthalmology, University of Pittsburgh School of Medicine, PA 15213, USA.
Exp Eye Res. 1993 Jun;56(6):635-48. doi: 10.1006/exer.1993.1081.
Corneal transparency is dependent on the size and arrangement of collagen fibrils within the corneal stroma. The corneal stroma is composed primarily of collagen type 1 fibrils and two proteoglycans: one with chondroitin/dermatan sulfate side-chains (decorin) and one with keratan sulfate side-chains (lumican). We investigated the effects of the corneal proteoglycans on corneal collagen fibrillogenesis, utilizing an in vitro assay for fibril formation. Collagen was extracted from bovine corneal stromas with 0.1 M acetic acid and monomers purified by NaCl precipitation. Decorin and lumican were extracted from bovine corneal stroma with either 0.7 M NaCl or 4 M guanidine HCl and purified by DEAE and Sepharose CL-4B chromatography. Decorin and lumican from both extracts inhibited the rate of collagen fibrillogenesis and the development of turbidity in fibrillogenesis samples. Furthermore, the core proteins of decorin and lumican were shown to be as effective as the intact proteoglycans in inhibiting fibrillogenesis. The decorin core protein isolated from the 0.7 M NaCl extract was determined to be a 20 kDa fragment which lacks the C-terminal half of the core protein. This fragment was approximately 1/36 as effective in inhibiting fibrillogenesis as intact decorin isolated from guanidine extracts. This suggests that the C-terminal half of the decorin core plays an important role in the interaction of this proteoglycan with collagen. Lumican extracted with 0.7 M NaCl was slightly smaller and was only one-sixth as effective in inhibiting collagen fibril formation as 4 M guanidine extracted lumican. Furthermore reduction and alkylation of lumican core protein abolished the inhibitory activity of the core protein on collagen fibrillogenesis. Electron microscopic examination indicated that fibrils formed in the presence of lumican and lumican core protein were significantly thinner than fibrils formed in the absence of proteoglycans. The results of these studies indicate that in addition to decorin, lumican retards corneal collagen fibrillogenesis and results in the formation of collagen fibrils which are significantly thinner than those formed in the absence of any proteoglycan. The inhibitory activity of lumican or decorin on collagen fibrillogenesis resides in he core proteins of these proteoglycans, not the glycosaminoglycan side chains, and that interaction of the lumican core protein with collagen appears to be dependent on the presence of disulfide bridges within the protein core.
角膜透明度取决于角膜基质中胶原纤维的大小和排列。角膜基质主要由I型胶原纤维和两种蛋白聚糖组成：一种带有硫酸软骨素/硫酸皮肤素侧链（核心蛋白聚糖），另一种带有硫酸角质素侧链（光蛋白聚糖）。我们利用一种体外纤维形成测定法，研究了角膜蛋白聚糖对角膜胶原纤维形成的影响。用0.1M乙酸从牛角膜基质中提取胶原，通过氯化钠沉淀纯化单体。用0.7M氯化钠或4M盐酸胍从牛角膜基质中提取核心蛋白聚糖和光蛋白聚糖，并用二乙氨基乙基纤维素和琼脂糖CL-4B柱层析纯化。两种提取物中的核心蛋白聚糖和光蛋白聚糖均抑制胶原纤维形成的速率以及纤维形成样品中浊度的发展。此外，核心蛋白聚糖和光蛋白聚糖的核心蛋白在抑制纤维形成方面与完整的蛋白聚糖一样有效。从0.7M氯化钠提取物中分离出的核心蛋白聚糖核心蛋白被确定为一个20kDa的片段，该片段缺少核心蛋白的C端一半。该片段在抑制纤维形成方面的效力约为从胍提取物中分离出的完整核心蛋白聚糖的1/36。这表明核心蛋白聚糖核心的C端一半在该蛋白聚糖与胶原的相互作用中起重要作用。用0.7M氯化钠提取的光蛋白聚糖略小，在抑制胶原纤维形成方面的效力仅为用4M盐酸胍提取的光蛋白聚糖的六分之一。此外，光蛋白聚糖核心蛋白的还原和烷基化消除了核心蛋白对胶原纤维形成的抑制活性。电子显微镜检查表明，在光蛋白聚糖和光蛋白聚糖核心蛋白存在下形成的纤维明显比在没有蛋白聚糖的情况下形成的纤维细。这些研究结果表明，除了核心蛋白聚糖外，光蛋白聚糖也会延缓角膜胶原纤维的形成，并导致形成比在没有任何蛋白聚糖的情况下形成的胶原纤维明显更细的胶原纤维。光蛋白聚糖或核心蛋白聚糖对胶原纤维形成的抑制活性存在于这些蛋白聚糖的核心蛋白中，而不是糖胺聚糖侧链中，并且光蛋白聚糖核心蛋白与胶原的相互作用似乎取决于蛋白核心内二硫键的存在。
